Optical biosensing of markers of mucosal inflammation.

We report the design and adaptation of iron/iron oxide nanoparticle-based optical nanobiosensors for enzymes or cytokine/chemokines that are established biomarkers of lung diseases. These biomarkers comprise ADAM33, granzyme B, MMP-8, neutrophil elastase, arginase, chemokine (C-C motif) ligand 20 and interleukin-6. The synthesis of nanobiosensors for these seven biomarkers, their calibration with commercially available enzymes and cytokines/chemokines, as well as their validation using bronchoalveolar lavage (BAL) obtained from a mouse model of TLR3-mediated inflammation are discussed here. Exhaled Breath Condensate (EBC) is a minimally invasive approach for sampling airway fluid in the diagnosis and management of various lung diseases in humans (e.g., asthma, COPD and viral infections). We report the proof-of-concept of using human EBC in conjunction with nanobiosensors for diagnosis/monitoring airway inflammation. These findings suggest that, with nanosensor technology, human EBC can be utilized as a liquid biopsy to monitor inflammation/remodeling in lung disease.

Early detection of pancreatic cancers in liquid biopsies by ultrasensitive fluorescence nanobiosensors.

Numerous proteases, such as matrix metalloproteinases (MMPs), cathepsins (CTS), and urokinase plasminogen activator (UpA), are dysfunctional (that is, over- or under-expressed) in solid tumors, when compared to healthy human subjects. This offers the opportunity to detect early tumors by liquid biopsies. This approach is of particular advantage for the early detection of pancreatic cancer, which is a "silent killer". We have developed fluorescence nanobiosensors for ultrasensitive (sub-femtomolar) arginase and protease detection, consisting of water-dispersible Fe/Fe3O4 core/shell nanoparticles and two tethered fluorescent dyes: TCPP (Tetrakis(4-carboxyphenyl)porphyrin) and cyanine 5.5. Upon posttranslational modification or enzymatic cleavage, the fluorescence of TCPP increases, which enables the detection of proteases at sub-femtomolar activities utilizing conventional plate readers. We have identified an enzymatic signature for the detection of pancreatic adenocarcinomas in serum, consisting of arginase, matrix metalloproteinase-1, -3, and - 9, cathepsin-B and -E, urokinase plasminogen activator, and neutrophil elastase, which is a potential game-changer.

A nanobiosensor for the detection of arginase activity.

A nanobiosensor for arginase detection was designed and synthesized. It features a central dopamine-coated iron/iron oxide nanoparticle to which sulfonated cyanine 7.0 is tethered via a stable amide bond. Cyanine 5.5 is linked to the N-terminal of the peptide sequence GRRRRRRRG. Arginine (R) reacts to ornithine (O) in the presence of arginase. Based on calibration with commercially obtained arginase II, the limit of detection (LOD) is picomolar. It is noteworthy that the nanobiosensor for arginase detection does not show a fluorescence increase when incubated with the enzyme NO-reductase, which also uses arginase as substrate, but is indicative of an inflammatory response by the host to cancer and infections. Arginase activity was determined in a syngeneic mouse model for aggressive breast cancer (4T1 tumors in BALB/c mice). It was found that the arginase activity is systemically enhanced, but especially pronounced in the active tumor regions.

Rationally designed peptide nanosponges for cell-based cancer therapy.

A novel type of supramolecular aggregate, named a "nanosponge" was synthesized through the interaction of novel supramolecular building blocks with trigonal geometry. The cholesterol-(K/D)nDEVDGC)3-trimaleimide unit consists of a trigonal maleimide linker to which homopeptides (either K or D) of variable lengths (n=5, 10, 15, 20) and a consensus sequence for executioner caspases (DEVDGC) are added via Michael addition. Upon mixing in aqueous buffer cholesterol-(K)nDEVDGC)3-trimaleimides and a 1:1 mixture of cholesterol-(K/D)nDEVDGC)3-trimaleimides form stable nanosponges, whereas cholesterol-(D)nDEVDGC)3-trimaleimide is unable to form supramolecular aggregates with itself. The structure of the novel nanosponges was investigated through explicit solvent and then coarse-grained molecular dynamics (MD) simulations. The nanosponges are between 80 nm and several micrometers in diameters and virtually non-toxic to monocyte/macrophage-like cells.

Nanoplatforms for highly sensitive fluorescence detection of cancer-related proteases.

Numerous proteases are known to be necessary for cancer development and progression including matrix metalloproteinases (MMPs), tissue serine proteases, and cathepsins. The goal of this research is to develop an Fe/Fe3O4 nanoparticle-based system for clinical diagnostics, which has the potential to measure the activity of cancer-associated proteases in biospecimens. Nanoparticle-based "light switches" for measuring protease activity consist of fluorescent cyanine dyes and porphyrins that are attached to Fe/Fe3O4 nanoparticles via consensus sequences. These consensus sequences can be cleaved in the presence of the correct protease, thus releasing a fluorescent dye from the Fe/Fe3O4 nanoparticle, resulting in highly sensitive (down to 1 × 10(-16) mol l(-1) for 12 proteases), selective, and fast nanoplatforms (required time: 60 min).

Channel blocking of MspA revisited.

Porin A from Mycobacterium smegmatis (MspA) is a highly stable, octameric channel protein, which acts as the main transporter of electrolytes across the cell membrane. MspA features a narrow, negatively charged constriction zone, allowing stable binding of various analytes thereby blocking the channel. Investigation of channel blocking of mycobacterial porins is of significance in developing alternate treatment methods for tuberculosis. The concept that ruthenium(II)quaterpyridinium complexes have the capability to act as efficient channel blockers for MspA and related porins, emerged after very high binding constants were measured by high-performance liquid chromatography and steady-state luminescence studies. Consequently, the interactions between the ruthenium(II) complex RuC2 molecules and MspA, leading to RuC2@MspA assemblies, have been studied utilizing time-resolved absorption/emission, atomic force microscopy, dynamic light scattering, ζ potential measurements, and isothermal titration calorimetry. The results obtained provide evidence for the formation of clusters/large aggregates of RuC2 and MspA. The results are of interest with respect to utilizing prospective channel blockers in porins. The combination of results from conceptually different techniques shed some light onto the chemical nature of MspA-channel blocker interactions thus contributing to the development of a paradigm for channel blocking.

Maleimide-functionalized photochromic spirodihydroindolizines.

Two photochromic spirodihydroindolizine/betaine systems for tethering to peptides and proteins via a maleimide function have been prepared. The absorption spectra of the betaines are in the red region of the visible spectrum and in the near-IR spectral domain, which are suitable energies of light for future in vivo applications. The half-times of cyclization have been determined for both DHI/betaine systems. The findings are consistent with a thermal barrier of varying size between the transoid and cisoid conformers of the betaines.

A cell-delivered and cell-activated SN38-dextran prodrug increases survival in a murine disseminated pancreatic cancer model.

Enzyme-activated prodrugs have been investigated and sought after as highly specific, low-side-effect treatments, especially for cancer therapy. Unfortunately, excellent targets for enzyme-activated therapy are rare. Here a system based on cell delivery that can carry both a prodrug and an activating enzyme to the cancer site is demonstrated. Raw264.7 cells (mouse monocyte/macrophage-like cells, Mo/Ma) are engineered to express intracellular rabbit carboxylesterase (InCE), which is a potent activator of the prodrug irinotecan to SN38. InCE expression is regulated by the TetOn® system, which silences the gene unless a tetracycline, such as doxycycline, is present. Concurrently, an irinotecan-like prodrug, which is conjugated to dextran and can be loaded into the cytoplasm of Mo/Ma, is synthesized. To test the system, a murine pancreatic cancer model is generated by intraperitoneal (i.p.) injection of Pan02 cells. Engineered Mo/Ma are loaded with the prodrug and are injected i.p. Two days later, doxycycline was given i.p. to activate InCE, which activated the prodrug. A survival study demonstrates that this system significantly increased survival in a murine pancreatic cancer model. Thus, for the first time, a prodrug/activating enzyme system, which is self-contained within tumor-homing cells and can prolong the life of i.p. pancreatic tumor bearing mice, is demonstrated.

Stem cell-based photodynamic therapy.

We have transfected murine neural stem cells (NSCs) and rat umbilical cord matrix-derived stem cells (RUCMSCs) with a plasmid expressing gaussia luciferase (gLuc). These cells are engineered to secrete the luciferase. We have used gLuc containing supernatant from culturing the NSCs to perform in vitro photodynamic therapy of murine melanoma cells (B16F10), and RUCMSCs to perform in vivo PDT of lung melanomas in C57BL/6 mice. The treatment system was comprised of aminolevulic acid as a prodrug for the synthesis of the photosensitizer protoporphyrin IX, gaussia luciferase, and its' substrate coelenterazine. A significant reduction of the number of live melanoma cells in vitro and a borderline significant retardation of tumour growth in vivo was observed after coelenterazine-mediated PDT.

A self-contained enzyme activating prodrug cytotherapy for preclinical melanoma.

Gene-directed enzyme prodrug therapy (GDEPT) has been investigated as a means of cancer treatment without affecting normal tissues. This system is based on the delivery of a suicide gene, a gene encoding an enzyme which is able to convert its substrate from non-toxic prodrug to cytotoxin. In this experiment, we have developed a targeted suicide gene therapeutic system that is completely contained within tumor-tropic cells and have tested this system for melanoma therapy in a preclinical model. First, we established double stable RAW264.7 monocyte/macrophage-like cells (Mo/Ma) containing a Tet-On® Advanced system for intracellular carboxylesterase (InCE) expression. Second, we loaded a prodrug into the delivery cells, double stable Mo/Ma. Third, we activated the enzyme system to convert the prodrug, irinotecan, to the cytotoxin, SN-38. Our double stable Mo/Ma homed to the lung melanomas after 1 day and successfully delivered the prodrug-activating enzyme/prodrug package to the tumors. We observed that our system significantly reduced tumor weights and numbers as targeted tumor therapy after activation of the InCE. Therefore, we propose that this system may be a useful targeted melanoma therapy system for pulmonary metastatic tumors with minimal side effects, particularly if it is combined with other treatments.

A/C magnetic hyperthermia of melanoma mediated by iron(0)/iron oxide core/shell magnetic nanoparticles: a mouse study.

BACKGROUND: There is renewed interest in magnetic hyperthermia as a treatment modality for cancer, especially when it is combined with other more traditional therapeutic approaches, such as the co-delivery of anticancer drugs or photodynamic therapy. METHODS: The influence of bimagnetic nanoparticles (MNPs) combined with short external alternating magnetic field (AMF) exposure on the growth of subcutaneous mouse melanomas (B16-F10) was evaluated. Bimagnetic Fe/Fe3O4 core/shell nanoparticles were designed for cancer targeting after intratumoral or intravenous administration. Their inorganic center was protected against rapid biocorrosion by organic dopamine-oligoethylene glycol ligands. TCPP (4-tetracarboxyphenyl porphyrin) units were attached to the dopamine-oligoethylene glycol ligands. RESULTS: The magnetic hyperthermia results obtained after intratumoral injection indicated that micromolar concentrations of iron given within the modified core-shell Fe/Fe3O4 nanoparticles caused a significant anti-tumor effect on murine B16-F10 melanoma with three short 10-minute AMF exposures. We also observed a decrease in tumor size after intravenous administration of the MNPs followed by three consecutive days of AMF exposure 24 hrs after the MNPs injection. CONCLUSIONS: These results indicate that intratumoral administration of surface modified MNPs can attenuate mouse melanoma after AMF exposure. Moreover, we have found that after intravenous administration of micromolar concentrations, these MNPs are capable of causing an anti-tumor effect in a mouse melanoma model after only a short AMF exposure time. This is a clear improvement to state of the art.

Development of a Gene Delivery System Composed of a Cell-Penetrating Peptide and a Nontoxic Polymer.

Major concerns have arisen with respect to using viral vectors for gene therapies. Collateral effects include cancer resistance, development of new cancers, and even systemic deaths. For this reason, researchers have focused on the alternative of using nonviral nanocarriers for gene therapy. In this study, a gene delivery nanocarrier was developed, comprising a cell-penetrating peptide called WTAS as a primary nanocarrier and a poly(ß-amino ester) (PBAE) polymer as a secondary nanocarrier. Here, the PBAE polymer is used to protect the WTAS peptide from early degradation while further facilitating the transportation into cells. WTAS is a peptide that penetrates cell nuclei within a few minutes after exposure, which makes it an ideal candidate to transport genetic materials. The PBAE-WTAS nanocarrier was assembled and tested against three cell lines (NSC, B16F10, and GL26). Cytotoxic studies demonstrated the relatively low toxicity of the PBAE-WTAS nanocarrier and PBAE-WTAS loaded with green fluorescent protein (GFP) plasmid DNA (pDNA@PBAE-WTAS) against all three cell lines. Cell transfection experiments were carried out using GL26 cells. These studies demonstrated a very high transfection rate of PBAE-WTAS loaded with GFP plasmid DNA, leading to virtually complete transfection (> 90%). In conclusion, we report a very promising gene delivery nanocarrier, which can be further modified to transport a variety of genetic materials for targeted therapy of multiple diseases.

Wharton's jelly-derived cells are a primitive stromal cell population.

Here, the literature was reviewed to evaluate whether a population of mesenchymal stromal cells derived from Wharton's jelly cells (WJCs) is a primitive stromal population. A clear case can be made for WJCs as a stromal population since they display the characteristics of MSCs as defined by the International Society for Cellular Therapy; for example, they grow as adherent cells with mesenchymal morphology, they are self-renewing, they express cell surface markers displayed by MSCs, and they may be differentiated into bone, cartilage, adipose, muscle, and neural cells. Like other stromal cells, WJCs support the expansion of other stem cells, such as hematopoietic stem cells, are well-tolerated by the immune system, and they have the ability to home to tumors. In contrast to bone marrow MSCs, WJCs have greater expansion capability, faster growth in vitro, and may synthesize different cytokines. WJCs are therapeutic in several different pre-clinical animal models of human disease such as neurodegenerative disease, cancer, heart disease, etc. The preclinical work suggests that the WJCs are therapeutic via trophic rescue and immune modulation. In summary, WJCs meet the definition of MSCs. Since WJCs expand faster and to a greater extent than adult-derived MSCs, these findings suggest that WJCs are a primitive stromal cell population with therapeutic potential. Further work is needed to determine whether WJCs engraft long-term and display self-renewal and multipotency in vivo and, as such, demonstrate whether Wharton's jelly cells are a true stem cell population.

Peptide Nanosponges Designed for the Delivery of Perillyl Alcohol to Glioma Cells.

Peptide nanosponges of low polydispersity are spontaneously formed from trigonal supramolecular building blocks in aqueous buffers, which feature cationic and/or anionic oligopeptides (n = 5-20) and a hydrophobic unit. In contrast to classical liposomes/vesicles, nanosponges feature interwoven hydrophilic and hydrophobic nanodomains and are readily taken up by mammalian cells. Perillyl alcohol is known to be a simple, but effective small molecule drug against glioma multiforme. However, its efficacy is limited by a poor bioavailability. In order to make perillyl alcohol bioavailable, two nanosponges consisting of 10 aspartates, to which perillyl alcohol is attached by means of an ester bond, and 20 lysines or arginines (type (D-POH)10K20 and (D-POH)10R20) were synthesized, purified, and characterized by dynamic light scattering (DLS) and atomic force microscopy (AFM). These nanosponges were then tested in cell cultures of murine glioma cells (GL26) and murine neural progenitor cells (NPC) because the latter was previously utilized in cell-based cancer therapy. The two nanosponges exhibited significantly different biophysical properties (size distribution and ζ potentials). Consequently, different efficacies in killing GL26 and NPC were observed in serum-containing culture media. The results from these experiments confirmed that the type (D-POH)10K20 nanosponge is a promising candidate for the (cell-mediated) cytotherapy of glioblastoma.

Developing a xenograft human tumor model in immunocompetent mice.

Animal models are essential to cancer research, but current xenograft models are limited in their utility especially due to the lack of an immune system. Here we demonstrate that a xenograft tumor model can be developed in immunocompetent mice by tolerizing murine fetuses to human tumor cells. A375 human melanoma cells were injected into day E14 fetuses and after birth mice were challenged with A375 cells to determine their ability to develop tumors. Intravenous injections of cells resulted in metastatic-like lung tumors, which were verified to be human in origin by immunohistochemistry and PCR. These results were replicated with several other human tumor types: BxPC3 (human pancreatic adenocarcinoma), MDA-MB-231 (human breast adenocarcinoma), M21 (human melanoma), and HeLa (human cervical adenocarcinoma). Development of an immunocompetent xenograft tumor model would allow the further elucidation of the interaction of the immune system with therapy in both preclinical research and patient derived xenografts.

Peptide nanosponges designed for rapid uptake by leukocytes and neural stem cells.

The structure of novel binary nanosponges consisting of (cholesterol-(K/D) n DEVDGC)3-trimaleimide units possessing a trigonal maleimide linker, to which either lysine (K)20 or aspartic acid (D)20 are tethered, has been elucidated by means of TEM. A high degree of agreement between these findings and structure predictions through explicit solvent and then coarse-grained molecular dynamics (MD) simulations has been found. Based on the nanosponges' structure and dynamics, caspase-6 mediated release of the model drug 5(6)-carboxyfluorescein has been demonstrated. Furthermore, the binary (DK20) nanosponges have been found to be virtually non-toxic in cultures of neural progenitor cells. It is of a special importance for the future development of cell-based therapies that DK20 nanosponges were taken up efficiently by leucocytes (WBC) in peripheral blood within 3 h of exposure. The percentage of live cells among the WBC was not significantly decreased by the DK20 nanosponges. In contrast to stem cell or leucocyte cell cultures, which have to be matched to the patient, autologous cells are optimal for cell-mediated therapy. Therefore, the nanosponges hold great promise for effective cell-based tumor targeting.

Associations between activity of arginase or matrix metalloproteinase-8 (MMP-8) and metritis in periparturient dairy cattle.

Metritis, a uterine disease caused by bacterial infection, is highly prevalent in dairy cattle after parturition. Uterine disease has negative effects on milk production and reproductive efficiency. Finding markers or indicators that can predict cows at greater risk for uterine disease could be beneficial to mitigating these deleterious effects. This study investigates the immune-derived enzymes arginase and matrix metalloproteinase-8 (MMP-8) as potential markers for development of metritis in dairy cows. In a retrospective matched case-control study, 53 lactating Holstein cows diagnosed with metritis were matched and paired to 53 lactating Holstein control cows. In addition to examining cows for diagnosis of metritis on d 4, 7, 10, and 14 after parturition, occurrence of retained fetal membranes, gender of the calf, and the event of a stillbirth were recorded. Blood samples were collected 7 ± 3 d before calving, on the day of calving, and 7 ± 3 d after calving and were assayed for activity of arginase and MMP-8. Associations between metritis and activity of arginase or MMP-8 were determined by conditional logistic regression at each individual sampling time point. An interaction between activity of arginase, before and on the day of parturition, and retained fetal membranes tended (P ≤ 0.13) to be associated with metritis. After parturition, activity of arginase and the interaction between activity of arginase and retained fetal membranes were not (P ≥ 0.22) associated with metritis. Activity of MMP-8 was not (P ≥ 0.20) associated with metritis in the periparturient period. Retained fetal membranes were associated with the odds of developing metritis. Activity of arginase before and at the time of parturition might be a potential marker for occurrence of metritis, especially in cows that develop retained fetal membranes. MMP-8 does not seem to be a potential indicator for metritis.

Synergy of Iron Chelators and Therapeutic Peptide Sequences Delivered via a Magnetic Nanocarrier.

Here, we report the synthesis, characterization, and efficacy study of Fe/Fe3O4-nanoparticles that were co-labeled with a tumor-homing and membrane-disrupting oligopeptide and the iron-chelator Dp44mT, which belongs to the group of the thiosemicarbazones. Dp44mT and the peptide sequence PLFAERL(D[KLAKLAKKLAKLAK])CGKRK were tethered to the surface of Fe/Fe3O4 core/shell nanoparticles by utilizing dopamine-anchors. The 26-mer contains two important sequences, which are the tumor targeting peptide CGKRK, and D[KLAKLAK]2, known to disrupt the mitochondrial cell walls and to initiate programmed cell death (apoptosis). It is noteworthy that Fe/Fe3O4 nanoparticles can also be used for MRI imaging purposes in live mammals. In a first step of this endeavor, the efficacy of this nanoplatform has been tested on the highly metastatic 4T1 breast cancer cell line. At the optimal ratio of PLFAERD[KLAKLAK]2CGKRK to Dp44mT of 1 to 3.2 at the surface of the dopamine-coated Fe/Fe3O4-nanocarrier, the IC50 value after 24 h of incubation was found to be 2.2 times lower for murine breast cancer cells (4T1) than for a murine fibroblast cell line used as control. Based on these encouraging results, the reported approach has the potential of leading to a new generation of nanoplatforms for cancer treatment with considerably enhanced selectivity towards tumor cells.

Stem cells in the umbilical cord.

Stem cells are the next frontier in medicine. Stem cells are thought to have great therapeutic and biotechnological potential. This will not only to replace damaged or dysfunctional cells, but also rescue them and/or deliver therapeutic proteins after they have been engineered to do so. Currently, ethical and scientific issues surround both embryonic and fetal stem cells and hinder their widespread implementation. In contrast, stem cells recovered postnatally from the umbilical cord, including the umbilical cord blood cells, amnion/placenta, umbilical cord vein, or umbilical cord matrix cells, are a readily available and inexpensive source of cells that are capable of forming many different cell types (i.e., they are "multipotent"). This review will focus on the umbilical cord-derived stem cells and compare those cells with adult bone marrow-derived mesenchymal stem cells.

Early breast cancer screening using iron/iron oxide-based nanoplatforms with sub-femtomolar limits of detection.

Proteases, including matrix metalloproteinases (MMPs), tissue serine proteases, and cathepsins (CTS) exhibit numerous functions in tumor biology. Solid tumors are characterized by changes in protease expression levels by tumor and surrounding tissue. Therefore, monitoring protease levels in tissue samples and liquid biopsies is a vital strategy for early cancer detection. Water-dispersable Fe/Fe3O4-core/shell based nanoplatforms for protease detection are capable of detecting protease activity down to sub-femtomolar limits of detection. They feature one dye (tetrakis(carboxyphenyl)porphyrin (TCPP)) that is tethered to the central nanoparticle by means of a protease-cleavable consensus sequence and a second dye (Cy 5.5) that is directly linked. Based on the protease activities of urokinase plasminogen activator (uPA), MMPs 1, 2, 3, 7, 9, and 13, as well as CTS B and L, human breast cancer can be detected at stage I by means of a simple serum test. By monitoring CTS B and L stage 0 detection may be achieved. This initial study, comprised of 46 breast cancer patients and 20 apparently healthy human subjects, demonstrates the feasibility of protease-activity-based liquid biopsies for early cancer diagnosis.